Erythropoietin (EPO), a 34 kd glycoprotein hormone produced primarily by the kidney, is the principal factor regulating erythropoiesis and plays a key role in stimulating erythrocyte formation in higher organisms. It serves as both mitogen and survival factor which acts on erythroid progenitors, such as erythroid colony-forming units (CFU-E), to promote these cells to proliferate and, possibly, to maturate. EPO is necessary for the replacement of erythrocytes which must occur on an ongoing basis because mature erythrocytes cannot grow or divide and have a limited lifespan. EPO can be used therapeutically for treatment of anemia.
The function of EPO in erythropoiesis is mediated by its cellular receptor (EPOR), a 507 amino acid polypeptide with a single transmembrane domain. Although the EPOR is expressed in a cell type-restricted fashion, i.e., in cells of erythroid lineage, its transcripts have also been etected in non-erythroid cell types such as megakaryocyte, ast cells, umbilical vein endothelial cells and cells with euronal characteristics.
The present invention relates to transgenic nonhuman ammals and their progeny, such as transgenic mice and their progeny, in which expression of erythropoietin is lacking (e.g., deleted); the endogenous (naturally occurring) erythropoietin receptor is deleted; the endogenous erythropoietin receptor is replaced by a heterologous erythropoietin receptor (i.e., one not present in the corresponding nontransgenic or wild type mammal); erythropoietin expression is lacking and the erythropoietin receptor is deleted; or erythropoietin expression is lacking and the erythropoietin receptor is replaced by a heterologous erythropoietin receptor. Transgenic mammals in which EPO expression is lacking or the EPOR is deleted (i.e., transgenic nonhuman animals carrying null mutations in the Epo gene or EpoR gene) are produced by knocking out (deleting or otherwise disabling) the endogenous EPO gene (Epo) or the endogenous EPO receptor gene (EpoR), respectively, using methods described herein and known to those in the field. Transgenic mammals in which the endogenous EPOR is replaced by a heterologous EPOR can carry a chimeric EPOR, such as a human/mouse chimeric EPOR, or an EPOR of another species, such as the human EPOR present in a mouse. Transgenic mammals in which EPO expression is lacking and the EPOR is lacking or is a heterologous EPOR are produced by breeding mice which lack EPO expression (EPO gene knockout mice) with mice in which, respectively, the EPOR is lacking or is a heterologous EPOR. For example, transgenic mice in which EPO expression is lacking and the mouse EPOR (mEPOR) is replaced with the human EPOR (hEPOR) are produced by breeding mice which are EPO gene knockouts with mice carrying the hEPOR gene. The transgenic mice or other nonhuman transgenic mammal can be heterozygous or homozygous for the knockout or deletion.
A further subject of the present invention is mammalian, nonhuman embryonic stem (ES) cells or cell lines in which the EPO gene or the EPOR gene has been knocked out (deleted or otherwise disabled) by the methods described herein and embryonic stem cell lines in which the EPOR gene has been replaced by a gene encoding a heterologous EPOR, which is a chimeric (e.g., human-mouse) EPOR or an EPOR which is not chimeric and is an EPOR from another animal (e.g., a gene encoding the hEPOR replacing the mEPOR).
The present invention also relates to nucleic acid constructs, particularly DNA constructs, useful for producing the transgenic nonhuman mammals, such as transgenic mice, described herein. It also relates to methods of identifying or designing agents or drugs, such as small molecules, which mimic the effects or activity of EPO. In a method of the present invention, a candidate agent is administered, for example, to a transgenic mouse and the effect of the agent on erythropoiesis is assessed. The transgenic mouse can, for example, lack EPO expression and carry a human/mouse chimeric EPOR in which the extracellular domain is the human EPOR extracellular domain, the transmembrane domain is either the mouse or the human EPOR transmembrane domain and the intracellular domain is the mouse EPOR intracellular domain. If, after the candidate agent is administered to the transgenic animal, the animal displays effects associated with administration of EPO (e.g., enhanced erythropoiesis, treatment of anemia), the agent is EPO-like in its effects; such an agent is referred to as an EPO mimic or EPO mimetic. An agent shown to have EPO-like effects in a transgenic animal in which the EPOR extracellular domain is human is particularly desirable because it has a demonstrated effect on the human EPOR. The present invention further relates to EPO mimics or mimetics and their use. Such EPO mimics can be used prophylactically or therapeutically in any context in which EPO can be used (e.g., to prevent or treat anemia, such as the anemia associated with renal failure).